Tickborne diseases are a serious health threat that has steadily risen, with reported cases more than doubling over the past 13 years. Lyme disease, or Lyme borreliosis, accounts for more than 80% of the reported cases of tickborne diseases in the US. It is estimated that over 300,000 new cases of Lyme disease occur per year in the US, with an estimated 85,000 cases a year in Europe. Our current work addresses the following areas in Lyme disease: development of new tests and biomarkers for infection, investigation of persistence of infection with B. burgdorferi in humans, and investigation of the role of immune response in Lyme disease and post treatment Lyme disease syndrome (PTLDS). One of the main problems in Lyme diagnosis has been the lack of highly specific and sensitive assays for B. burgdorferi and the lack of a test that could be used to assess response to therapy. Such assays should greatly facilitate the accurate diagnosis of Lyme disease and assessment of response to therapy in individual patients. Animal studies have shown that B. burgdorferi may persist in animals after antibiotic therapy and can be detected by using the natural tick vector (Ixodes scapularis) to acquire the organism through feeding. Whether this occurs in humans is unknown. We have completed a phase I study to investigate the utility of this approach for identifying persistence of B. burgdorferi in treated human Lyme disease, and are performing the phase 2 study. We have helped develop the Tick-Borne Disease Serochip (TBD-Serochip), in collaboration with Dr. Rafal Tokarz and Dr. Ian Lipkin (Columbia University). The TBD-Serochip targets 8 major tick-borne pathogens, including Anaplasma phagocytophilum, Babesia microti, B. burgdorferi, Borrelia miyamotoi, Ehrlichia chaffeensis, Rickettsia rickettsii, Heartland virus and Powassan virus. Each assay contains approximately 170,000 12-mer linear peptides that tile along the protein sequence of the major antigens from each agent with 11 amino acid overlap. Our initial investigation has that the assay can be employed to discriminate individual antibody responses in patients with tickborne diseases and examine the interplay of agents on disease manifestation and progression. We are examining the antibody response to immunogenically dominant antigens of B. burgdorferi in PTLDS patients and controls. Further investigation of the anti-borrelia immune response may help in elucidating the pathogenic mechanism of PTLDS and yield important information for future approaches to diagnosis and treatment. We have characterized the gene expression patterns in skin biopsies from individuals with EM, to learn about the human host response at the site of the infection. Calprotectin is a potent antimicrobial that inhibits the growth of pathogens by tightly binding transition metals such as Mn and Zn, thereby preventing their uptake and utilization by invading microbes. Together with Dr. Culotta (Johns Hopkins University), we have shown that calprotectin is produced by both epidermal keratinocytes and in immune cells infiltrating the dermis in erythema migrans (the rash of Lyme disease). Calprotectin inhibited B. burgdorferis growth in culture, but the mechanism does not involve the classical withholding of metal nutrients, while requiring physical association of calprotectin and the bacteria. B. burgdorferi is one of these few extracellular pathogens capable of establishing persistent infection in mammals. In collaboration with Dr. Utpal Pals at UMD-CP, we have investigated the role of B. burgdorferi surface protein BBA57 in early and late infection. We showed that BBA57 function is critical for early infection but largely redundant for later stages of spirochetal persistence, either in mammals or in ticks. BBA57 facilitates early infection by regulating host IFN responses and suppressing host microbicidal activities. Loss of BBA57 results in near clearance of pathogens at the inoculum site but triggers nonheritable adaptive changes that allows BBA57-deficient mutants to compensate for gene loss during the later disseminated phase of infection. Understanding the mechanisms of immune evasion is critical for the development of novel therapeutic approaches to combat Lyme borreliosis. We are also investigating the immunological response in neuroborreliosis. Results from these studies give us a window into the fundamental biology of the infection.